Substance with fibrinolytic activity and method of manufacturing the same

ABSTRACT

This invention relates to a new fibrinolytic substance and the method of manufacturing the same which can be singly isolated from venom of venomous snakes belonging to the family of Trimeresurus taxonomically by means of molecular sieve chromatography and ion exchange chromatography. This substance has the action of dissolving fibrin in mammals by activating plasminogen and its clinical use a fibrinolytic agent is promising.

DESCRIPTION

1. Technical Field

This invention relates to a new substance which is obtained from venomof venomous snakes belonging to the family of trimeresurus taxonomicallyand which has fibrinolytic activity to mammals, and a method ofmanufacturing the same and its applications in medicine.

2. Background Art

In general, animal toxin such as venom of snakes is known to involvevarious proteinic toxin factors, some of which may endorse the presenceof materials showing various biochemical actions such as, for example,hemorrhagic toxin, neurotoxin, hemolytic constituent, hemopexisconstituent, etc. Recent years have witnessed biochemical studies ofthese animal toxins which are well in progress.

Taking note of the action of the venom of Trimeresurus which is fearedto have very often caused fatalities to human life and cattle, thepresent inventor has long been engaged in studying the action of proteintoxin, and their effects on the blood coagulation system, fibrinolyticsystem and plastocyte function. He has discovered that a substanceobtained by a special treatment of crude venom of Trimeresurus has astrong fibrinolytic activity and, as the result of furtherinvestigation, he has succeeded in isolating such substance singly andmoreover, found that it has an extensive range of clinical uses as afibrinolytic agent.

DISCLOSURE OF INVENTION

The principal object of the present invention resides in providing a newsingly isolated substance, a method of manufacturing the same and itsapplications in medicine, said new substance being obtained byfractionating a solution of crude venom of Trimeresurus by means ofso-called molecular sieve chromatography as the main procedure using astationary phase of material having a molecular sieve effect, incombination with ion exchange chromatography using a stationary phase ofion exchange material so as to collect fractions of the largest activityof fibrinolysis each time in the aforesaid chromatographic procedure.The present invention will be explained in detail centering on preferredembodiments hereinafter.

Crude venom of Trimeresurus for use as the material in the presentinvention is defined and is to be understood as toxin which can beobtained from venomous snakes belonging to the family of TrimeresurusCrotalidae by classification, for example, Trimeresurus flavoviridis,Trimeresurus tokarensis, Trimeresurus okinavensis, Trimeresurusmucrosquamatus, Trimeresurus elegans, Trimeresurus stejnegsi,Trimeresurus tinkami, Trimeresurus moticola, and Trimeresurus gracilis.

A freezedried powder of crude venom obtained from one or more of thesevenomous snakes, is dissolved in a suitable solvent, for example,distilled water, physiological saline or a buffer solution of pH withinthe range of 6 to 8. This solution is fractionated by the so-calledmolecular sieve chromatography using a stationary phase of materialhaving a molecular sieve effect. For example, a suitable gel filtrationagent such as Sephadex (G-75 or G-100) (Trade name) or Biogel (Tradename) is used as a column filler in which said solution is adsorbed andpermeated and subsequently, the resulting solution thus adsorbed andpermeated is eluted in a buffer solution of suitable electrolyte of pH,about 6-8, thereby enabling it to be fractionated into 4 fractions, P-I,P-II, P-III and P-IV. Each of these fractions is tested by the standardfibrin plate method so that the fraction P-II can be ensured to have anactivity of fibrinolysis.

This fraction, P-II is subjected to dialysis in distilled water and theinternal solution after dialysis is freezedried. Subsequently, thefraction P-II thus freezedried is dissolved in a buffer solution of pHaround 6 such as sodium acetate buffer solution and the resultingsolution is fractionated by ion exchange chromatography. Namely, it isadsorbed in a column filled with CM cellulose as a cation exchange bodyat low temperature and the portion thus adsorbed is eluted in a buffersolution by slowing increasing salt concentration in the buffer solutionso that a portion having the largest activity of fibrinolysis can becollected, and freezedried. Since this fraction is found to contain oneor more kinds of impurities by SDS electrophoresis, it is furthersubjected to a refining step by molecular sieve chromatography.Specifically the fraction obtained by ion exchange chromatography whichis then freezedried, is dissolved in a buffer solution of pH within therange of 6-8 and the resulting solution is adsorbed in a column filledwith Sephadex (G-50) and the portion thus adsorbed is eluted in asimilar buffer solution so that a substance having an activity offibrinolysis can be isolated as the objective of the present invention.This substance is found to be a single chemical compound by SDSelectrophoresis.

Sephadex G-50 is a kind of cross-linked dextran permitting protein of amolecular weight, less than 10,000, to be diffused in its gel particles,having a cross-linking extent of water regain, 5 g/g (dry particles) andSephadex G-75 is capable of fractionating protein of a molecular weight,less than 40,000, and having a cross-linking extent, water regain 7.5g/g.

Although Sephadex and Biogel have been exemplified as gel filtrationagents of polysaccharide hereinbefore, other gel filtration agentshaving such molecular sieve effect can also be used in a like manner. Atthe same time, the ion exchange material is not restricted to CMcellulose but it is also possible to select some other suitable materialwhich is compatible with the pH of the effluent in use. The preferredbuffer solutions can be enumerated such as sodium citrate buffersolution, acetic acid buffer solution, phosphoric acid buffer solutionand boric acid buffer solution.

The above mentioned operations are possible at ordinary temperature(room temperature) but lower temperature is better, for example, 5° C.is found most suitable.

Although the above mentioned operations of crude venom have been carriedout in the order of molecular sieve chromatography using Sephadex(G-100), subsequently, ion exchange chromatography and molecular sievechromatography using Sephadex (G-50) as a further refining step, suchorder is only one example. In other words, ion exchange chromatographymay be carried out first or last as a refining step so that the samepurpose may be achieved.

The substance with fibrinolytic activity thus obtained may be dissolved,as is, in physiological saline to produce an injection. It may also betreated by the conventional procedure for making it suitable for use asa medicine according to necessity.

The physical properties of the fibrinolytic substance of the presentinvention are that it is soluble in water, its sugar content can berecognized by the Anthrone method and it is a glycoprotein having itsmaximal absorption spectrum at 620-640 nm.

According to gel filtration with Sephadex (G-50) and discelectrophoresis, its molecular weight is presumed to be 28,000-32,000and its total nitrogen content is 12.4±1%. This substance shows itsabsorption spectrum with ultraviolet ray at the maximal wavelength 278nm. An infrared absorption spectrum of this substance by the KBr tabletmethod is shown in FIG. 3 and its maximal absorption of large scale islocated in the neighbourhood of wavenumber, 3400, 1435, and 1350 and itsmaximal absorption of medium scale in the neighbourhood of wavenumber,1640, 1100 and 925 and its maximal absorption of small scale in theneighbourhood of wavenumber, 780 respectively.

The composition of amino acids of this substance by an amino-acidanalyser is shown in Table 1 and FIG. 4.

                  TABLE 1                                                         ______________________________________                                               Amino acid                                                                             N mol                                                         ______________________________________                                               Aspartic acid                                                                          15.366                                                               Threonine                                                                              7.132                                                                Serine   5.578                                                                Glutamic acid                                                                          9.797                                                                Glycine  9.475                                                                Alanine  7.668                                                                Cystine  5.454                                                                Valine   7.422                                                                Methionine                                                                             2.936                                                                Isoleucine                                                                             4.836                                                                Leucine  7.215                                                                Tyrosine 8.923                                                                Phenylalanine                                                                          2.895                                                                Lysine   17.729                                                               Ammonia  13.278                                                               Histidine                                                                              1.793                                                                Arginine 3.674                                                         ______________________________________                                    

Tests of pharmacological effect of this substance were made byfibrinolysis of fibrin plate and by Chandler Loop test (Chandler, A. B.Lab. Invest., 7, 110 (1958): Conner, W. E. & Poole, J. C. F. Quart. J.Exp. Physiol. 40 1 (1961).). The first mentioned method has a measuringprinciple which consists in measuring the dissolved area of a fibrinplate as a substrate due to the fibrinolytic activity of the testmaterial thus enabling the activity to be observed. The second methodconsists in measuring the ability of the substance for dissolvingartificial thrombus formed in a loop tube.

The fibrin plate for use in the present invention is one which does notcontain plasminogen (precursor of plasmin).

First of all, in the case of causing the substance of the presentinvention only to act on the fibrin plate, no dissolving effect can beseen and therefore, the substance has no plasmin action. On the otherhand, in the case of causing a mixed solution of purified plasminogenand the present substance to act on the fibrin plate, there is noted anoutstanding fibrinolytic action as compared with a control solutioncontaining plasminogen only. (See FIG. 2). For the sake of confirmation,when the present substance is allowed to act on the standard fibrinplate, its apparently dissolving tendency can be recognized andtherefore, the mode of action can be determined as an activator likeaction.

Also by the Chandler Loop method, the present substance showed a markedeffect of dissolving thrombus.

As shown in the above mentioned pharmacological test results, the newsubstance having such fibrinolytic activity as is disclosed in thepresent application is expected to develop the undermentioned clinicaleffects due to the fact that it can activate plasminogen to becomeplasmin capable of dissolving fibrin.

In general, fibrin converted from fibrinogen by an enzymatic action isknown to be one of the serious causes of thrombosis and embolism.

Owing to the aforesaid action of this substance, the inventive substanceis expected to develop a prophylactic and therapeutic effect onperipheral arteriovenous thrombosis, pulmonary embolism, coronaryinfarction, myocardinal infarction, cerebral blood vessel infarction,thrombosis of retinal artery and vein, hemorrhage of the vitreous body,hyphema and other such diseases. Moreover, in combination with acarcinostatic agent, the present substance is expected to showsynergistic action for the therapy of cancer. In addition, the presentsubstance may be useful as anticoagulants in blood transfusion and anagent for preventing embolus formation at a suture line in vasotomy orfor prolonging the physical functions of arteriovenous shunt in blooddialysis.

In the case of using the present substance in therapy, it may beadministered by intravenous injection or local irrigation as the casemay be.

While its doses are different depending on the kind of disease and itsadministration, and also, age, body weight of patients and seriousnessof symptoms, a sufficient therapeutic effect can be obtained with asingle dose of 0.1-80 mg for adults in most cases. Experimental examplesof its pharmacology are shown as follows.

EXPERIMENT 1

Acute toxicity of the present substance was tested in groups of malemice of ddN (body weight 21±2 g), each group consisting of 10 mice, byintravenous injection to show LD₅₀, 20 mg/kg after 72 hours, bycalculation of Litchfield & Wilcoxon's method.

EXPERIMENT 2

For the purpose of determining the fibrinolytic action of the presentsubstance, when a sample of protein concentration, 450 μg/ml is appliedto a fibrin plate (plasminogen free fibrin plate manufactured by KowaCo., Ltd., ditto hereinafter), it does not dissolve the fibrin plate.Therefore, the present substance has no plasmin activity.

EXPERIMENT 3

A physiological salt solution of purified plasminogen (manufactured byKABI Company), 5 cu/ml was prepared. 50 μl of this solution was mixedwith 100 μl of each of solutions consisting of the present substancedissolved in a Tris-HCl 0.05 M buffer solution (pH 7.4) to produce saidsolutions of protein concentration, 40, 120, 370, 950 and 1900 μg/mlrespectively. Then 5 μl of the resulting mixed solution was poured ontoa hole formed on a fibrin plate to cause its action thereon.

After leaving it alone at 37° C. for 20 hours, the dissolved area of thefibrin plate was measured, thus determining an activator action of thepresent substance. The fibrin plate used in this experiment is noted toshow a dissolved ring with a clear border corresponding to thefibrinolytic action of the substance. The diameter of this ring isrectilinear proportion to a logarithm of fibrinolytic activity(concentration) so that the activity as an activator can be observed bymeasuring such diameter. The results of this measurement are shown inTable 2 and FIG. 2.

In Table 2, the activity of this substance as an activator prove to bean outstanding one as compared with a control (plasminogen only) andthis activity as an activator turns out to be dose-dependent.

                  TABLE 2                                                         ______________________________________                                               Frequency of test                                                      Protein                                                                       concentration                                                                          1         2      3      4    Average                                 ______________________________________                                         40 (μg/ml)                                                                         5.60      5.45   5.40   5.55 5.50                                     120     6.40      6.15   6.10   6.25 6.23                                     370     7.70      7.55   7.40   7.35 7.50                                     950     8.20      8.30   8.10   8.15 8.19                                    1900     9.70      9.20   9.70   9.50 9.53                                    Control  0         0      0      0    0                                       ______________________________________                                    

EXPERIMENT 4

The present substance was dissolved in Tris buffer solution in anicecooled test tube to produce solutions of protein concentration, 1125,225, and 150 μg/ml respectively. 0.3 ml of each solution was mixed with0.05 ml of a solution of purified human plasminogen, 15 CTA unit/ml and0.1 ml of a solution of thrombin-Ca (50 unit--0.1 M/ml). To theresulting mixed solution was added 0.05 ml of a solution of purifiedhuman fibrinogen, 2.6% and the resulting solution was placed in a warmbath of 37° C. to form fibrin clot, and the time adding said fibrinogensolution till the fibrin clot dissolved was measured. The results areshown in Table 3.

At the same time, in place of this solution, a control usingphysiological saline was left alone for more than 6 hours but the fibrinclot could not be dissolved.

                  TABLE 3                                                         ______________________________________                                        Protein concentration                                                                          Time of dissolving                                           of present substance                                                                           fibrin clot                                                  (μg/ml)       (minutes)                                                    ______________________________________                                        1125              70                                                          225              143                                                          150              307                                                          ______________________________________                                    

EXPERIMENT 5

The substance was dissolved in Tris-HCl buffer solution to produce asolution of protein concentration, 370 μg/ml and was caused to act on astandard fibrin plate. After 20 hours, the diameter of the dissolvedring was measured. The results are shown in Table 4.

                  TABLE 4                                                         ______________________________________                                        Poured amount of                                                                              Diameter of dis-                                              substance       solved ring                                                   (μl)         (mm)                                                          ______________________________________                                         5              7.65                                                          10              8.49                                                          20              9.32                                                          ______________________________________                                    

EXPERIMENT 6

1 ml of human flesh blood with the addition of citric acid and 0.1 ml of0.25 M CaCl₂ solution were put in a plastic tube, internal diameter 3 mmand length 270 mm. Both ends of the tube were joined together to form aloop which was then rotated for 30 minutes at 37° C. to complete theformation of thrombus. The substance of this invention was dissolved infresh human blood to produce solutions of protein concentration 0.24mg/ml and 1.4 mg/ml respectively. These solutions were added to saidthrombus and the loop thus treated was rotated for 4 hours at 37° C.after which the remaining thrombus was withdrawn from the tube andhomogenized with distilled water so as to be dissolved. The amount ofhemoglobin was measured at wavelength 540 nm by colorimetry to determinethe fibrinolytic action of the substance. The results are shown in Table5.

                  TABLE 5                                                         ______________________________________                                        Protein concentration                                                                         Percentage of dissolving                                      of substance    thrombus                                                      (mg/ml)         (%)                                                           ______________________________________                                        0.24            11                                                            1.4             60                                                            ______________________________________                                    

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 shows an ultraviolet absorption spectrum of the substance.

FIG. 2 shows a graph of the action of the substance on plasminogen.

FIG. 3 shows an infrared absorption spectrum of the substance.

FIG. 4 shows a chart of amino acid composition of the substance by anaminoacid analyser. In FIG. 4, there are shown the respective peaks ofamino acids as follows.

    ______________________________________                                        1        Aspartic acid 2      Threonine                                       3        Serine        4      Glutamic acid                                   5        Proline       6      Glycine                                         7        Alanine       8      Cystine                                         9        Valine        10     Methionine                                      11       Isoleucine    12     Leucine                                         13       Tyrosine      14     Phenylalanine                                   15       Lysine        16     Ammonia                                         17       Histidine     18     Arginine                                        ______________________________________                                    

BEST MODE FOR CARRYING OUT THE INVENTION

For the purpose of clarifying the present invention further, a preferredembodiment for manufacture of the present substance will be describedhereinbelow.

(EXAMPLE)

3 g of freezedried crude venom of Trimeresurus flavoviridis Hallowell isdissolved in 15 ml of 0.02 M boric acid buffer solution (pH 7.5) atordinary temperature. The resulting solution is poured into a column,5×90 cm filled with Sephadex (G-75) and the resulting solution is elutedin 0.02 M boric acid buffer solution (pH 7.5) at an eluting speed, 60ml/hr at 5° C. and a fraction having fibrinolytic activity is collectedand subjected to dialysis in distilled water. Then the dialysed internalsolution of dialysis is freezedried. 100 mg of this freezedried materialis dissolved in 40 ml of 0.05 M sodium acetate buffer solution (pH 6.0)and the resulting solution is adsorbed in a column, 1.5×40 cm filledwith CM cellulose and eluted by slowly increasing salt concentration insaid sodium acetate buffer solution till it becomes 0.3 M and a fractionhaving the largest activity of fibrinolysis is collected andfreezedried. Subsequently, 50 mg of this fraction is dissolved in 2 mlof 0.02 M boric acid buffer solution (pH 7.5) and the resulting solutionis poured into a column, 35×90 cm filled with Sephadex (G-50) and elutedat 5° C. in 0.02 M boric acid buffer solution and then a fraction of thelargest activity of fibrinolysis is collected. This fraction proves tobe a uniform material by SDS electrophoresis. The percentage of thesubstance is 3%.

I claim:
 1. A method of manufacturing a substance having the activity ofa plasminogen activator, which comprises subjecting a solution of venomof Trimeresursur to multiple chromatography consisting essentially of acombination of:providing a first molecular sieve chromatography usingSephadex G-75 or G-100 as a stationary phase; thereafter providing asecond molecular sieve chromatography using Sephadex G-50 as astationary phase; and at a time before or after or intermediate saidfirst and second chromatographies, carrying out an ion exchangechromatography using a cation exchange body as a stationary phase;fractionating each protein-containing solution eluted in turn duringeach stage of said first, second and ion exchange chromatography using abuffer solution of electrolyte with pH 6-8 as a mobile phase; confirmingthe plasminogen activator activity of each of the fractionated portions;collecting the fractionated portions having the largest plasminogenactivator activity; dializing said largest activity fractionatedportions in distilled water; and subjecting said largest activityportions to an operation of separation by subsequent chromatography,thereby singly isolating a substance with plasminogen activatoractivity.
 2. A manufacturing method, as claimed in claim 1, whichcomprises using carboxylmethyl cellulose as said cation exchange body.3. A manufacturing method, as claimed in claim 1, which comprises usingone of a process for measuring an area of fibrinolysis of a fibrin plateor a Chandler Loop Test in confirming the activity of plasmin activator.4. A manufacturing method, as claimed in claim 1, wherein said buffersolution of electrolyte with pH 6-8 is a solution selected from thegroup consisting of sodium citrate buffer solution, acetic acid buffersolution, phosphoric acid buffer solutions and boric acid buffersolutions.
 5. A method of manufacturing a substance with the activity ofa plasminogen activator, which comprises:subjecting the venom ofTrimeresursur flavoviridis to column chromatography using one ofSephadex G75 and G-100 as a stationary phase; confirming the activity ofa plasminogen activator in each fractionated portion obtained by thevenom thus treated being eluted in boric acid buffer solution,collecting its portion having the strongest activity of a plasminogenactivator and, after dialyzing these collected portions in distilledwater, freeze-drying said collected portions; subsequently subjectingsaid collected and freezedried portions to column chromatography usingcarboxymethyl cellulose as a stationary phase so as to be eluted by theuse of a sodium acetate buffer solution; collecting the fractions of thelast-mentioned chromatography having the strongest activity of aplasminogen activator; freeze-drying the last-mentioned fractions andfurther subjecting said last-mentioned fractions thus freeze-dried, tocolumn chromatography using Sephadex G-50 as a stationary phase so as tobe eluted in boric acid buffer solution; and collecting fractions of theSephadex G-50 chromatography having the strongest activity of aplasminogen activator.
 6. A manufacturing method, as claimed in claim 5,which comprises using one of a process for measuring the dissolved areaof a standard fibrin plate or a Chandler Loop Test for confirming theactivity of a plasminogen activator.
 7. A substance with a strongplasminogen activator activity obtained by subjecting a solution ofvenom of Trimeresurus to multiple chromatography consisting essentiallyof a combination of: providing a first molecular sieve chromatographyusing Sephadex G-75 or G-100; thereafter providing a second molecularsieve chromatography using Sephadex G-50 as a stationary phase; and, ata time before or after or intermediate said first and secondchromatographies, carrying out ion exchange chromatography using acation exchange body as a stationary phase; fractionating eachprotein-containing solution eluted in turn during each stage of saidfirst second and ion exchange chromatographies using a buffer solutionof electrolyte with pH 6-8 as a mobile phase; confirming the plasminogenactivator activity of each of the fractionated portions; collecting thefractionated portions having the largest plasminogen activator activitydialyzing said largest activity fractionated portions in distilledwater; and subjecting said largest activity portions to an operation ofseparation by subsequent chromatography, thereby singly isolating asubstance with plasminogen activator activity, which substance has itsmaximal absorption spectrum in the proximity of ultraviolet ray, 278 nmand infrared ray, around 3400 cm⁻¹ (large scale), 1640 cm⁻¹ (mediumscale), 14355 cm⁻¹ (large), 1350 cm⁻¹ (large), 1250⁻¹ (small scale),1100 cm⁻¹ (medium), 1025 cm⁻¹ (small), 925 cm⁻¹ (medium) and 780 cm⁻¹(small) respectively and having a molecular weight within the range of28,000-35,000 and observed to have a glycoprotein having a total mountof nitrogen, 12.4±1%, being soluble in water and capable of operating aplasminogen activator, the amino acids of the substance being present ina relative N mol quantity as follows,

    ______________________________________                                               Aspartic acid                                                                          15.366                                                               Threonine                                                                              7.132                                                                Serine   5.578                                                                Glutamic acid                                                                          9.797                                                                Glycine  9.475                                                                Alanine  7.668                                                                Cystine  5.454                                                                Valine   7.422                                                                Methionine                                                                             2.936                                                                Isoleucine                                                                             4.836                                                                Leucine  7.215                                                                Tyrosine 8.923                                                                Phenylalanine                                                                          2.895                                                                Lysine   17.729                                                               Ammonia  13.278                                                               Histidine                                                                              1.793                                                                Arginine 3.674.                                                        ______________________________________                                    